T regulatory cells mediate immunosuppresion by adenosine in peripheral blood, sentinel lymph node and TILs from melanoma patients.

T regulatory cells (Tregs), involved in tumour tolerance, can generate Adenosine by CD39/CD73 surface enzymes, which identify four Tregs subsets: CD39+CD73- nTregs, CD39+CD73+ iTregs, CD39-CD73+ oTregs and CD39-CD73- xTregs. In melanoma patients, increased Tregs levels are detected in peripheral blood (PB), sentinel lymph node (SLN) and tumour infiltrating lymphocytes (TILs), but Adenosine role was not investigated yet. We examined total Tregs and Adenosine subsets in PB, SLN and TILs from melanoma patients (n = 32) and PB from healthy donors (HD; n = 10) by flow cytometry. Total Tregs significantly increased in stage III-IV patients PB, in SLN and TILs, as compared to HD/stage I-II patients. Tregs subsets analyses showed that: 1) PB nTregs significantly increased in SLN and decreased in TILs; 2) iTregs significantly increased in stage III-IV patients PB and further significantly increased in SLN and TILs; 3) PB oTregs and xTregs significantly decreased in SLN and TILs. Patients clinical features did not significantly influence total Tregs, except SLN excision order. Results confirmed Tregs role in melanoma progression and indicate Adenosine generation as a novel escape mechanism, being nTregs and iTregs increased in PB/SLN/TILs.

Mixed acute leukemia with genotypic lineage switch: a case report.

Morphologically well classifiable leukemias can reveal a mixed phenotype. A case of acute myeloid leukemia (CD13, CD33, CD14, CD11b) which at presentation showed a co-expression of B-lymphoid markers (CD19, CD10, CD20), at the time of the first relapse revealed a morphologic, phenotypic and genotypic switch of the blasts to a purely lymphoid form. Analysis of the immunoglobulin (Ig) H chain locus and of the T-cell receptor (TCR) genes showed at diagnosis a germline configuration of the IgH, TCR beta and tau genes, and a deletion of the TCR delta gene at the second chromosome. At relapse, monoclonal rearrangements of the IgH, TCR tau, and TCR delta were detected. At a subsequent relapse, the blasts re-expressed myeloid morphologic features and myeloid-associated antigens, while they retained the same rearranged configuration of the IgH and TCR beta and delta genes. The TCR delta gene configuration, which links each phase of the disease, may represent an early pathogenetic event and makes the emergence of a second malignancy unlikely. Each phenotypic change occurred after anti-myeloid and anti-lymphoid oriented chemotherapy. The close correlation between the progressive acquisition of different phenotypes and the switch at the genomic level represent the peculiar features of this unusual case.

Megakaryocyte-like increase in ploidy of Friend's erythroleukemia cells induced to endoreplication by colcemid.

Previous work from this laboratory has shown that Friend's murine erythroleukemia cells (MELCs) express some bio-chemical traits of the megakaryocytic lineage. The supposed mixed erythroid/megakaryocytic nature of these cells has been investigated further by challenging MELCs with the antimicrotubule agent colcemid. This compound, at the concentration of 40 nM, was found to induce a striking arrest of cell growth without significant effects on viability. At the same time, the bulk of treated MELCs underwent a large increase in size to contain, after 3 days, as much as 4 times more proteins and 5 times more DNA than controls. As shown by high rates of 3H-thymidine incorporation, increase in DNA content was the result of active synthesis without completion of intervening mitosis according to a process that closely resembled endoreplication. Eventually, colcemidinduced MELCs presented multilobed nuclei and were arranged into discrete ploidy groups containing up to 16 N levels of DNA. Moreover, upon colcemid addition, MELCs initiated a polyploid response that was shown to continue, even in the absence of the inducer, to yield cells that became strongly positive for acetylcholinesterase (AChE) in the late stages of culture. These effects were compatible with a colcemid-induced commitment of MELCs to megakaryocyte differentiation, for which these cells seemed to be definitely programmed. The expression of megakaryocyte features in MELCs provided further evidence for the bipotentiality (erythroid/megakaryocytic) of this model.

Severe hypoxia enhances the formation of erythroid bursts from human cord blood cells and the maintenance of BFU-E in vitro.

Incubation in severe hypoxia (1% oxygen) increased the number of erythroid bursts generated from full-term CD34+, or premature mononucleated, human cord blood (CB) cells, in semisolid cultures containing stem cell factor (SCF), interleukin (IL)-3 and erythropoietin (EPO). Severe hypoxia also enhanced the maintenance of erythroid burst-forming units (BFU-E) in CB cell liquid cultures. These positive effects of hypoxia on the maintenance and cloning efficiency of BFU-E did not extend to the other progenitors assayed. Hypoxia, on the other hand, markedly reduced the size and level of hemoglobinization of bursts and, in liquid cultures, suppressed the growth factor-stimulated numerical increase in BFU-E and inhibited the expression of CD36, a marker of erythroid colony-forming units and maturing erythroid precursors. However, when transferred to clonal assays incubated in air, cells from liquid cultures incubated in hypoxia or in air generated fully expanded and hemoglobinized bursts, suggesting that in hypoxia the clonogenic potential of BFU-E was maintained and the development of erythroid clones reversibly inhibited. These results indicate that hypoxia inversely regulates two subsequent phases of erythropoiesis, i.e., it enhances the maintenance of BFU-E and the early development of erythroid clones but inhibits the terminal expansion and maturation of these clones. The cloning of CB cells selected for CD34 positivity, when compared with that of the total population of mononucleated CB cells, revealed that the early development of erythroid bursts was either hypoxia-enhanced or hypoxia-insensitive, reflecting the existence of two different types of BFU-E. Hypoxia-enhanced BFU-E are relatively immature, are maintained in hypoxia but not in air, and account for a large part of CD34+ BFU-E and for a high percentage of the BFU-E in premature CB. Hypoxia-insensitive BFU-E are mostly CD34- and are largely predominant in full-term CB, and most probably correspond to a more mature type of BFU-E.

Erythropoietin upregulates the expression of its own receptor in TF-1 cell line.

In the erythroleukemia cell line TF-1, recombinant human erythropoietin (rHEpo), but not c-kit ligand, enhanced the number of cells expressing the erythropoietin receptor (EpoR), as measured by flow-cytometric analysis of binding of the biotin-labeled Epo. Moreover, 125I-Epo binding and Scatchard analyses, indicated that TF-1 cells, maintained in standard conditions with IL-3, and those stimulated with c-kit ligand, bear a single class of EpoR. On the other hand, cells cultured in the presence of rHEpo had a higher number of receptors than IL-3 or c-kit ligand-stimulated cells, and had two binding sites with different affinities for the ligand. EpoR mRNA expression was higher in cells exposed to rHEpo than in IL-3 or c-kit-stimulated cells. This difference may have been dependent on either a higher level of transcription or an increased stability of mRNA. The observed changes of EpoR in rHEpo-stimulated TF-1 cell line could cooperate, together with the alteration of the gene (3' end deletion), in the occurrence of the erythroleukemic process. Changes induced in EpoR by rHEpo were not accompanied by an increase in the expression of glycophorin A or globin chain mRNAs. This may suggest that rHEpo is unable to induce erythroid differentiation in TF-1 cells. The results also indicate that this cell line could be a model for the investigation of the role of transcription factor(s) in the expression of EpoR, and for the study of the mechanism(s) underlying the changes in the number and affinity of the cell receptors.

Continuous-infusion cyclophosphamide in combination with teniposide and dexamethasone in refractory myeloma.

Forty-three consecutive patients with refractory myeloma, median age 60, received monthly courses of teniposide 30 mg/m2 i.v. on days 1-2, dexamethasone 40 mg i.v. on days 1-7 and cyclophosphamide 200 mg/m2 by continuous i.v. infusion for seven days. Major response (decrease > 50% of M-protein) was achieved in 18 of 37 evaluable patients and minor response in 9, with an overall response rate of 73%. Response was irrespective of disease status, time from diagnosis and previous treatments, while beta 2 microglobulin > 6 mg/l was a powerful prognostic factor. All patients experienced transient granulocytopenia but extramedullary toxicity was negligible. Median survival of the whole group is 20 months, with 74% of responding patients projected to be alive at 30 months. In refractory myeloma cyclophosphamide appears to be more active when given by continuous infusion.

Low-dose cytosine arabinoside in patients with acute myeloid leukemia not eligible for standard chemotherapy.

The results of treatment with low dose cytosine arabinoside (LDARA-C) in 131 AML patients ineligible for standard regimens were analyzed retrospectively. Eighty-seven were previously untreated, 25 were refractory to conventional chemotherapy and 19 were relapsed patients. The median age was 66 years (15-84). An antecedent hematological disorder (AHD) was documented in half of the patients. Overall, 22 (17%) achieved complete remission, 14 (11%) partial remission, 77 (59%) had resistant leukemia and 18 died during induction. Median disease free survival was 57 weeks and median survival, for the 87 previously untreated patients, was 22.5 weeks. The prognostic value of initial parameters was analyzed for response. Bone marrow cellularity was the only significant factor. We observed 33% vs 81% (p < 0.01) of responses in patients with normo-hypercellular and hypocellular marrow, respectively. Accordingly, there was a trend to more responses in patients with leukocyte counts of less than 10 x 10(9)/L. M4-M5 FAB subtypes were frequently resistant to LDARA-C, resulting in a lower response rate compared to M0-M2 (18% vs 32%). Other parameters, including age, sex, hemoglobin, platelet count, AHD and fever at diagnosis, had no prognostic value. Our findings suggest that LDARA-C may be an effective treatment for some patients who are not eligible for first line conventional chemotherapy. However, this schedule is not advised in patients with monocytic leukemia or those with an hypercellular marrow.

Natural fluorescence of white blood cells: spectroscopic and imaging study.

Autofluorescence has been proved to be an intrinsic parameter of biological substrates that may aid in both the characterization of the physiological state and the discrimination of pathological from normal conditions of cells, tissues and organs. In this work, the fluorescence properties of human white blood cells have been studied in suspension and on single cells at microscopy. The results indicate that suspensions of agranulocytes and granulocytes differ in the amplitude of the fluorescence signal on excitation at wavelengths in the range 250-370 nm. The differences are particularly enhanced when excitation is performed in the 250-265 nm range. Microspectrofluorometric analysis, performed on single cells, allows several leukocyte families to be characterized. Lymphocytes, monocytes, neutrophils and eosinophils can be distinguished according to the intensity and spectral shape of the autofluorescence emission in the visible range from 440 to 580 nm. Both the nature and extent of the differences change when the excitation wavelength is moved from 366 to 436 nm. Differences in the intrinsic metabolic engagement, rather than in the cell dimensions, seem to be responsible for the differences observed between the leukocyte populations. The results identify interesting perspectives for autofluorescence as a discriminating parameter in the differential counting of human white blood cells.

Long-term suppression of EAE relapses by pharmacological impairment of epitope spreading.

BACKGROUND AND PURPOSE: Immune events sustaining dendritic cell (DC)-dependent epitope spreading (ES) are of key relevance to the development of relapses during multiple sclerosis (MS). Although no drugs are currently available to target ES, its inhibition would represent a major advancement in MS therapy. Inhibitors of the enzyme PARP-1 afford protection in animal models of MS, such as experimental autoimmune encephalomyelitis (EAE). These drugs epigenetically impair antigen presentation by DCs, but whether these drugs affect ES is unknown. Here, we investigated whether short-term treatments with these compounds would impair ES, thereby preventing EAE relapses. EXPERIMENTAL APPROACH: We used a model of relapsing EAE in SJL mice and also adopted in vivo and ex vivo models of DC-dependent T-cell polarization. The effect of PARP-1 inhibitors on ES was evaluated at the humoral and cellular level. KEY RESULTS: Short-term treatments with PARP-1 inhibitors during the acute phase of relapsing EAE of mice induced, at later times, more tolerogenic DCs, increased numbers of Treg cells and impairment of ES at the humoral and cellular level. These effects are followed by long-lasting reduction of relapse severity and incidence, although drug treatment had been discontinued for several weeks. PARP-1 inhibitors also induced tolerogenic DCs and increased Treg cells number and function in a model of ovalbumin immunization. CONCLUSIONS AND IMPLICATIONS: Our data emphasize the therapeutic potential of PARP-1 inhibitors in the treatment of relapsing-remitting MS and additional ES-driven autoimmune disorders.

Reticulated platelets predict cardiovascular death in acute coronary syndrome patients. Insights from the AMI-Florence 2 Study.

Reticulated platelets (RP) are newly-formed platelets with a greater mass, a residual amount of RNA and an increased prothrombotic potential. No studies investigating the association between RP and the risk of cardiovascular death in acute coronary syndrome (ACS) patients are available. In the frame of the AMI-Florence 2 study, we investigated RP in 229 (154 M/ 75 F) ACS patients (125 ST-elevation myocardial infarction [STEMI]; 104 Non-STEMI/Unstable Angina). RP were measured by using the Sysmex XE-2100 haematology analyzer and were expressed as the percentage of RP out of the total optical platelet count (immature platelet fraction; IPF) and as the percentage of RP highly fluorescent (H-IPF). At one-year follow-up, 22 out of 229 patients (9.6%) died from cardiovascular causes. Higher values of IPF (p=0.05) and H-IPF (p=0.006) were detected in dead compared to alive patients. A receiver operating characteristics curve analysis identified IPF ≥3.3% and H-IPF ≥0.9% as optimal cut-off values to predict cardiovascular death. At the multivariate model adjusted for the Global Registry of Acute Coronary Events (GRACE) risk score, the association between RP and cardiovascular death remained significant for both IPF [OR (95%CI) : 4.15 (1.24-13.91) p=0.02] and H-IPF [OR (95%CI): H-IPF 5.03 (1.38-18.38) p=0.01]. In conclusion, RP are independent predictors of cardiovascular death and may be useful in improving risk stratification for ACS patients. Future prospective studies to evaluate the role of RP in determining cardiovascular events are warranted.

Use of CFDA-SE for evaluating the in vitro proliferation pattern of human mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitors retaining the capability to undergo multilineage differentiation, mostly towards all the mesodermal cellular lineages. MSC growing under standard conditions are composed of two main subpopulations with a characteristic distribution in the morphologic flow cytometric scatter: RS (recycling stem) cells (small, agranular) and m (mature) MSC (large, moderately granular cells). METHODS: MSC obtained from BM of healthy donors and expanded in culture were characterized by evaluating both the expression of conventional markers and differentiation potential. We used CFSE, a lipophilic dye that is taken up by cell membranes, to investigate separately the proliferative activity of RS cells and mMSC subsets. RESULTS: With flow cytometric analysis, RS cells and mMSC showed nearly the same immunophenotypic pattern, even if a significantly smaller percentage of RS cells expressed some of the classic mesenchymal Ag. The RS cell fraction was confirmed to have a higher proliferative potential and such a feature was particularly evident under certain culture conditions. DISCUSSION: CFSE has been shown as a reliable method for studying the proliferative activity of MSC subpopulations identified by flow cytometric analysis. The acquisition parameter strategy is crucial for the accuracy of the analysis.

Growth inhibition and differentiation of human breast cancer cells by the PAFR antagonist WEB-2086.

WEB-2086 -- an antagonist of platelet-activating factor receptor (PAFR) with known anti-inflammatory, antiangiogenic and antileukaemic properties -- also proved to inhibit the proliferation in human solid tumour cell lines of different histology, and with much higher efficacy than in normal fibroblasts. A detailed analysis of WEB-2086 anticancer activity was then performed focusing on breast adenocarcinoma MCF-7 and MDA-MB-231 cells. WEB-2086-treated cells, either expressing (MCF-7) or unexpressing (MDA-MB-231) the oestrogen receptor (ER)alpha, underwent a dose-dependent growth arrest (IC(50)=0.65+/-0.09 and 0.41+/-0.07 mM, respectively) and accumulation in G(0)-G(1) phase. WEB-2086 also induced morphological and functional changes typical of mature mammary phenotype including (i) cell enlargement and massive neutral lipid deposition (best accomplished in MCF-7 cells); (ii) decrease in motility and active cathepsin D levels (mainly observed in highly invasive MDA-MB-231 cells). The expression of ERalpha was neither increased nor reactivated in treated MCF-7 or MDA-MB-231 cells, respectively. WEB-2086-induced differentiation in breast cancer cells involved the upregulation of PTEN, a key tumour suppressor protein opposing tumorigenesis, and was apparently independent of p53, PAFR, peripheral benzodiazepine receptor and ERalpha status. Overall, WEB-2086 can be proposed as an effective antiproliferative and differentiative agent with interesting translational opportunities to treat breast cancers in support to conventional chemotherapy.

Protective effects of the PARP-1 inhibitor PJ34 in hypoxic-reoxygenated cardiomyoblasts.

To clarify the role of poly(ADP-ribose)polymerase-1 (PARP-1) in myocardial ischemia-reperfusion injury, we explored some effects of PJ34, a highly specific inhibitor of this enzyme, in hypoxic-reoxygenated (HR) H9c2 cardiomyoblasts. Compared to the control, HR cells showed signs of oxidative stress, marked PARP-1 activation, NAD(+) and ATP depletion and impaired mitochondrial activity. HR cardiomyoblasts were affected by both necrosis and apoptosis, the latter involving the nuclear translocation of apoptosis-inducing factor. In HR cardiomyoblasts treated with PJ34, oxidative stress and PARP-1 activity were decreased, and NAD(+) and ATP depletion, as well as mitochondrial impairment, were attenuated. Above all, PJ34 treatment improved the survival of HR cells; not only was necrosis significantly diminished, but apoptosis was also reduced and shifted from a caspase-independent to a caspase-dependent pathway. These results suggest that PARP-1 modulation by a selective inhibitor such as PJ34 may represent a promising approach to limit myocardial damage due to post-ischemic reperfusion.

VEGF inhibition and cytotoxic effect of aplidin in leukemia cell lines and cells from acute myeloid leukemia.

BACKGROUND: Aplidine (APL) is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans that is under clinical phase II development. In contrast to the lack of bone marrow toxicity reported in phase I/II studies, it has been shown to induce cytotoxicity at very low concentration against lymphoblastic leukemia blast, as well as having an impact in the vascular endothelial growth factor (VEGF)/VEGF receptor 1 loop. PATIENTS AND METHODS: To confirm these findings we investigated APL-related VEGF inhibition and its cytotoxic effect on myeloid leukemic cells lines (K-562, HEL and HL60) and fresh leukemia blasts derived from 30 patients with acute myeloid leukemia (AML). The conventional active 4-demetoxi-daunorubicin (idarubicin; IDA) was included as a positive control. RESULTS: APL was found to be significantly (P<0.001) more active than IDA in obtaining 50% growth-inhibition in K-562, HEL and HL60 cell lines. Results obtained with AML blast cells were super imposible. ID(50) ranged from 0.024 to 0.610 microM for IDA (0.200+/-0.176) and from 0.001 to 0.108 microM for APL (0.020+/-0.031). Annexin V tests and cell cycle analysis performed on cell lines confirmed the stronger citotoxic capability of APL as apoptotic inducer and as a G(1) blocker. The inhibitory effects of APL on VEGF release and secretion have been confirmed by ELISA tests performed on HEL: the VEGF concentration in cell surnatant was reduced from 169 to 36 pg/ml after 24 h of exposure to a pharmacological concentration of APL. CONCLUSIONS: APL harbors a strong in vitro antileukemic activity at a concentration achievable in patients at non-myelotoxic doses. Our data also support the notion of an impact on VEGF secretion. Clinical studies with this new marine-derived compound in relapsed/resistant leukemia are underway.